1. Field of the Invention
The capability of manipulating DNA sequences encoding a polypeptide has greatly expanded the ability to synthesize a wide variety of polypeptides of commercial and physiological interest, as well as to modify the capability and functioning of cells. Unicellular microorganisms can be used to prepare a wide variety of polypeptides which naturally occur in mammals. In employing the unicellular microorganisms, it is desirable to enhance the production of the product of interest as compared to the total protein produced by the cell. In this manner, the cost of the product can be substantially reduced in relation to the cost of maintenance and growth of the microorganisms. Therefore, there have been many approaches in devising ways to enhance expression of the product of interest.
Of the unicellular microorganisms, yeast provides many advantages for the production of polypeptides employing recombinant or hybrid DNA technology. There already exists a large body of developed technology for the use of yeast in fermentation and for the production of many chemicals of commercial interest. Thus, conditions, growth, media, and methods of purification are already available.
Yeast are safe organisms and have been industrially used for an extended period of time, so that the handling and disposal is well established. Yeast are eukaryotic, rather than prokaryotic. Therefore, yeast are more likely to have analogous mechanisms for expression and more efficiently recognize the codons of a gene associated with a higher mammalian source.
In employing yeast for the production of polypeptides exogenous to the yeast, it is desirable that maximized production of the polypeptides of interest is achieved subject to limitations on the viability of the yeast. One technique which has encouraged higher yields of polypeptides of interest in a foreign host has been the use of multicopy plasmids. Multicopy plasmids suffer from a number of deficiencies, not least of which is that one must produce a large amount of DNA and the substances associated with the maintenance and replication of such DNA due to the multiple copies of the genes on the multicopy plasmid. Thus a substantial amount of the host's energy is dedicated to the production of products of no interest to the host or the manufacturer.
Another technique is to employ a highly efficient promoter, particularly a host promoter which is associated with the natural production of a polypeptide which is present as a high percentage of the total protein of the host or provides a high transcription turnover rate.
A third technique is to provide a way which amplifies the gene of interest. This technique has involved the use of a gene and its regulatory system which responds to stress by multiplication of the gene. It is found that by joining a foreign gene as a flanking region to the stress amplifiable gene, multiple repeats occur which will include both genes.
2. Description of the Prior Art
Kagi and Nodberg, Eds. (1979) Metallothionein (Birkhauser, Basel) and Beach and Palmiter, Proc. Natl. Acad. Sci., U.S.A. (1981) 78:210-214 describe metallothioneins generally. Brenes -Pomales et al, Nature (1955) 176: 841-842 describe copper sensitive and copper resistant yeast strains. These polymorphisms were then studied extensitively as markers in investigating gene conversion. Mortimer and Fogel (1974) In; Mechanisms in Recombination, R. Grell, Ed. (Plenum Press, New York) Pages 263-275, is illustrative of these studies. See also PCT application Nos. U.S. 81/00239 and U.S. 81/00240.